Human cancer-related gene, its encoded products and applications

ABSTRACT

The invention discloses a human cancer-related gene, LAPTM4B, its encoded products and their applications thereof. This human cancer-related gene provided by this invention comprises one of the following nucleotide sequences: (1) SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 6, or SEQ ID No: 8 in the sequence listings; (2) Polynucleotides that encode the protein sequences of SEQ ID No: 4, SEQ ID No: 5, or SEQ ID No: 7 in the sequence listings; (3) DNA sequences having above 90% homology with the DNA sequences specified by SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 6, or SEQ ID No: 8 in the sequence listings, and these DNA sequences encode the proteins with the same or similar functions. This invention enables the developments of new anti-cancer approaches and new anti-cancer medicines. It would create a significant impact on human society.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/019,297, filedFeb. 1, 2011, incorporated by reference herein in its entirety, which isa divisional of U.S. Ser. No. 10/540,539, filed Oct. 4, 2006, now U.S.Pat. No. 7,910,711, which is a national stage of PCT/CN2003/01109, filedDec. 24, 2003, which claims the priority benefit of Chinese ApplicationNo. 02158110.X, filed Dec. 24, 2002, and Chinese Application No.03109786.3, filed Apr. 21, 2003.

SEQUENCE LISTING

The following application contains a sequence listing in computerreadable format (CRF), submitted as a text file in ASCII format entitled“SequenceListing,” created on May 3, 2016, as 22 KB. The content of theCRF is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a human cancer-related gene, itsencoded products and their applications in genetic engineering andprotein engineering areas, as well as in medical diagnosis andtreatment.

BACKGROUND OF THE INVENTION

Cancer is the major health problem threatening human lives.Hepatocellular carcinoma (HCC) is one of the most serious among cancerdiseases. It is reported that the new cases of primary hepatocellularcarcinoma exceeds over one million worldwide each year. 70% of the newcases occur in Asia, and about 40-45% of the worldwide new cases occurin China. The total number of new hepatocellular carcinoma cases everyyear in China is about 450,000, and the number is increasing, especiallyin those between ages 20-60. The high incidence, difficulty in earlydiagnosis, fast growing rate, high reoccurrence, and the high mortalityrate make HCC a most malignant cancer. Most HCC patients have alreadyprogressed to the intermediate stage or late stage when diagnosed, andthey can only survive for 3-6 months if without a proper treatment.

To elucidate the mechanism underlying cancerogenesis would help forcancer prevention, diagnosis and treatment. Early diagnosis is crucialfor raising the curative rate and reducing the mortality. Currently usedHCC-diagnostic marker, the serum AFP, has 30% of negative results in HCCpatients, while some benign liver disease can cause a significantincrease of AFP level in serum, creating some difficulty in differentialdiagnosis. It has been found that the hepatocarcinogenesis is related toindividual hereditary susceptibility. Individuals with different geneticbackgrounds possess different handling capability toward environmentalcarcinogens. This leads to different risk of suffering from cancer forindividuals. It is the various genotypes and the genetic diversity thatcause the different genetic susceptibility for cancerogenesis.

Cancer is essentially a cellular hereditary disease. Although a greatnumber of cancer-related genes have been discovered, the mechanisms ofthe cancerogenesis and the development remain to be elucidated. Theknown oncogenes can be divided into five categories according to thecellular localization and function of their encoded proteins: I. genesthat encode growth factors, including sis, int-2, hst, fgf-5; II. genesthat encode growth factor receptors, including erbB, erbB-2, fins, met,ros, and others; III. genes that encode signal transduction molecules incytoplasm, including abl, src, ras, raf, yes, fgr, fes, lck, mos, andothers; IV. genes that encode regulatory molecules for cellproliferation and apoptosis, including bcl-1, bcl-2 and others; and V.genes that encode the nuclear DNA-binding proteins (transcriptionfactors), such as myc, myb, fos, jun, B-lym, ski, ets, rel and others.It has been demonstrated that ras, src, myc, met and p53 etc. are thegenes closely associated with HCC.

SUMMARY OF THE INVENTION

This invention provides a novel human cancer-related gene and itsencoded products.

This novel human cancer-related gene provided by this invention isdesignated as LAPTM4B. It comprises one of the following nucleotidesequences:

1. The human cancer-related gene comprises one of the followingnucleotide sequences:

-   -   1). SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 or SEQ ID No: 6 in        the sequence listings;    -   2). Polynucleotides that encode the protein sequences of SEQ ID        No: 4, SEQ ID No: 5, or SEQ ID No: 7 in the sequence listings;    -   3). DNA sequences having more than 90% homology with the DNA        sequences defined by SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 or        SEQ ID No: 6 in the sequence listings. These DNA sequences can        encode proteins having the same or similar functions.

SEQ ID No: 1 in the sequence listings mentioned above contains 954bases. It is an intact open reading frame. SEQ ID No: 1 has two startingsites, one is the base at 1-3 site at 5′ terminal, and another is thebase at 274-276 site at 5′ terminal. Two complete cDNAs in SEQ ID No: 1have two alternative tailing signals. When 5′ terminal in SEQ ID No: 1is extended outward by 85 bases, and 3′ terminal is extended outward by401 bases, SEQ ID No: 2 in the sequence listings is obtained. This genecontains 1440 bases. When 5′ terminal in SEQ ID No: 1 is extendedoutward by 85 bases, and 3′ terminal is extended outward 1130 bases, SEQID No: 3 in the sequence listings is obtained. This gene is consisted of2169 bases. LAPTM4B gene localizes on chromosome 8q22.1.

In the sequence listings, SEQ ID No: 6 is the allelic gene of SEQ ID No:1, consisting of 2264 bases. Its open reading frame starts from 17 to1129 base. This sequence contains two tandemly arranged 19 bp DNAsegments, the sequence of which is gcttgg agctccagca gct. These 19 bpDNA segments localized in nt 124-nt 161 in SEQ ID No: 6.

The human cancer related LAPTM4B protein possesses the amino acidsequence of 4, and/or 5, and/or 7. Or it consists of the sequence 4,and/or the sequence 5, and/or the sequence 7 after one or several aminoacid residues are replaced, deleted, or added. However, the abovealtered sequence 4, and/or the sequence 5, and/or the sequence 7 stillhave the same or similar activity to the unchanged sequence 4, and/orthe sequence 5, and/or the sequence 7.

Sequence 4 in the sequence listings consists of 317 amino acid residuesencoded by the whole sequence of SEQ ID No: 1. Its molecular mass is 35kDa and the putative isoelectric point is 9.05. Sequence 5 in thesequence listings contains 226 amino acid residues encoded by thesegment of bases from 274th to 954th in the SEQ ID No: 1. Its molecularmass is 24 kDa, and the putative isoelectric point is 4.65. The sequence7 in the sequence listings is a protein containing 370 amino acidresidues.

LAPTM4B gene is widely expressed at different levels in sixteen normaltissues. Its transcriptive expression is very high in testis, cardiacmuscle, and skeletal muscle, moderate in ovary, kidney, and pancreas,low in liver, spleen, small intestine, large intestine, and thymus, andis very low in lung and peripheral blood cells. In eight fetal tissues,the expression is high in heart, skeletal muscle, and kidney. In fetallivers, it is slightly higher than that in adult livers. However, itsexpression in some cancerous tissues is significantly upregulated. Forinstance, the Northern Blot analysis indicates that the transcriptionlevel in 87.3% (48/55) human hepatocellular carcinoma tissues issignificantly higher than that in fetal livers and normal livers (FIG.1-A). In situ hybridization (FIG. 2-A), immunohistochemistry (FIG. 2-B),and immunocytochemistry (FIG. 2-C) also indicate that LAPTM4B geneexpression is especially high in hepatocellular carcinoma tissues, whileits expression is relatively low in paired non-cancerous liver tissues(FIGS. 2-A and 2-B). Among the five cell lines from hepatoma tissuestested, all except for HLE, SMMC-7721, QGY-7701, BEL7402 and HG116 areexpressed highly (FIG. 1-B and FIG. 2-C). It is important that highlyover expressed protein product in hepatocellular carcinoma tissue andhepatocellular carcinoma cell line is mainly SEQ ID No: 4 LAPTM4B-35,while SEQ ID No: 5 LAPTM4B-24 only shows a slightly up regulation in itsexpression level. This results in a remarkable increase in the ratio ofLAPTM4B-35 to LAPTM4B-24 proteins in the hepatocellular carcinoma tissue(FIG. 2-B). Although the expressions of LAPTM4B-35 and LAPTM4B-24 areslightly increased in the paired non-cancerous tissue, their ratio isthe same as that in the normal liver (See Table 1). This is probably aprecancerous sign of hepatocellular carcinoma. In addition, theexpression levels of mRNA and the protein of LAPTM4B gene is negativelycorrelated with the differentiation of the hepatocellular carcinomatissue. The hepatocellular carcinoma tissues in low differentiation areexpressed highly, while the ones in high differentiation are expressedrelatively low (FIG. 1-C). The Western Blot and the immunohistochemicalmethod are used to determine the relationship of LAPTM4B gene with othercancers. The results indicate that LAPTM4B-35 protein expression is upregulated in some epithelium derived cancerous tissues and cell lines,such as stomach cancer, breast cancer, highly metastatic human lungcancer, and prostate cancer (FIG. 11). Moreover, in syngeneic human lungcancer and prostate cancer cell lines, LAPTM4B-35 expression is greatlyup regulated in cells of high metastasis potential compared with thoseof low metastasis potential. But in cell lines of human melanoma, eitherfrom in situ or metastatic cancer, it is not clearly expressed. AlthoughLAPTM4B-35 is expressed at a low level in liver tissues of adult ratsand mice, its expression is not obviously up regulated in either mouseascetically grown hepatocellular carcinoma or in the regenerated ratliver under a normal proliferation status.

TABLE 1 Expression ratio of LAPTM4B-35 to LAPTM4B-24 in hepatocellularcarcinoma tissue, paired non-cancerous liver tissue and normal livertissue HCC PNL NL LAPTM4B-35 13.32 ± 1.98 4.58 ± 1.31 2.78 ± 0.11LAPTM4B-24  3.59 ± 1.78 1.76 ± 1.24 1.00 ± 0.02 LAPTM4B-35/ 3.71 2.602.78 LAPTM4B-24 (Ratio) P < 0.01 HCC vs. PNL and NL

LAPTM4B proteins in SEQ ID No: 4, SEQ ID No: 5 and SEQ ID No: 7 havefour fragments of membrane-spanning sequences, one N-glycosylation site,a typical lysosome and endosome targeting signals in the cytoplasmicregion. They all belong to the protein superfamily of thetetratransmembrane proteins. However, they have various number ofphosphorylation sites. The experiment shows that SEQ ID No: 4 LAPTM4B-35can form a complex in plasma membrane with the integrin α6β1 (Singlespecific receptor of laminin in the extracellular matrix) and theepidermal growth factor receptor (EGFR) (FIGS. 14-A, B, and C). Thiscomplex is colocalized in cell plasma membrane. It is possible thatLAPTM4B-35 may integrate in the plasma membrane the proliferationsignals from both extracellular matrix and the growth factor. This canfurther elucidate molecular mechanism of the anchorage-dependent cellgrowth of normal eukaryotic cells, i.e. the eukaryotic cell growth needsnot only the stimulating signal from the growth factor, but also adefinite stimulating signal from extracellular matrix. It represents abreak through in understanding the regulation mechanism of the cellproliferation. Experiments demonstrate that tyrosine group (Tyr₂₈₅) inthe cytoplasmic region of LAPTM4B protein C terminal can bephosphorylated (FIG. 15-A). When the cell is attaching onto the lamininsubstrate, its phosphorylation is increased sharply (FIG. 15-A) and canbe completely inhibited by LAPTM4B-EC2-pAb antibody (FIG. 15-B), but thenon-correlated antibody does not show any inhibitory effect (FIG. 15-C).After the phosphorylation, Tyr₂₈₅ forms a site to bind with the SH2domain of intracellular signal molecules. In the meantime, N terminaland C terminal sequences of LAPTM4B contain Pro-rich domains and bindingsites of the typical SH3 I domain. The above results indicate that SEQID No: 4 LAPTM4B-35 protein may be an important docking protein forsignal transduction, or an organizer of the special microdomain in theplasma membrane. It can recruit related signal molecules from bothinside and outside of the cells to complete the signal transduction forcell proliferation, differentiation, and apoptosis. Experimental resultsshow that the transfection of mouse NIH3T3 cells and HLE humanhepatocellular carcinoma cells by cDNA in SEQ ID No: 4 produces stabletransfected and LAPTM4B-35 over expressed NIH3T3-AE and HLE-AE celllines. The growth curves (FIG. 4), the incorporation of 3H-TdR (FIG. 5),and the cell numbers in S phase of cell cycle (FIG. 6) all demonstratethat the rate of cell proliferation is greatly increased. Moreover, theproliferation of transfected cells shows less dependence on the growthfactor in serum, and the transfected cells can form large colonies insoft agar. Inoculation of NIH mouse with NIH3T3-AE cells can form amoderate malignant fibrosarcoma (FIG. 7), indicating the over expressionof LAPTM4B-35 induces out of control of the cell proliferation. Alsomigration capability of the HLE-AE cells is strengthened and itscapability to invade the Matrigel is remarkably enhanced, indicatingthat the over expression of LAPTM4B-35 accelerates the development ofcell malignant phenotype. On the contrary, the cDNA of SEQ ID No: 5 (Anencoding sequence where 91 amino acids in the N terminal of LAPTM4B-35is truncated) transfected mouse BHK, NIH3T3, and HLE hepatocellularcarcinoma cell lines cannot survive for a long time. The result showsthat the 91 amino acid sequence on the N terminal of SEQ ID No: 4LAPTM4B-35 protein play a crucial role in regulating cell proliferation.LAPTM4B-35 protein and LAPTM4B-24 protein have reciprocal,antagonistical functions in cell proliferation and survival. Theoverexpression of LAPTM4B-35 accelerates cellular malignanttransformation, while the overexpression of LAPTM4B-24 facilitates thecell death. Their expression equilibration and regulation are pivotal tothe carcinogenesis and progression of malignant cancer. LAPTM4B gene maybelong to the proto-oncogene family. In cancer treatment, inhibiting SEQID No: 4 LAPTM4B-35 expression and strengthening SEQ ID No: 5 LAPTM4B-24expression may suppress the growth of hepatocellular carcinoma andreverse its malignant phenotype or progressively slow down itsdevelopment. Furthermore, the overexpression of LAPTM4B-35 also promotesupregulation of the cell cycle regulators, such as cyclin D1 (FIG. 13-A)and cyclin E (FIG. 13-B), and also the over expression of someproto-oncogenes, such as c-Myc (FIG. 13-C), c-Jun (FIG. 13-D), and c-Fos(FIG. 13-E) etc.

The monoclonal and polyclonal antibodies for SEQ ID No: 4 LAPTM4B-35protein epitopes, such as polyclonal LAPTM4B-EC2₂₃₂₋₂₄₁-pAb for SEQ IDNo: 4 LAPTM4B-35 in the secondary extracellular region, polyclonalantibodies (LAPTM4B-N₁₋₉₉-pAb and LAPTM4B-N₂₈₋₃₇-pAb) for SEQ ID No: 4LAPTM4B-35 N terminal sequence, and monoclonal antibodies for LAPTM4Bare important in studying the effects of LAPTM4B-35 and LAPTM4B-24 incancer diagnosis and treatment (FIGS. 2, 3, 8, 11, 12, 14, 15). Forexample, LAPTM4B-EC2₂₃₂₋₂₄₁-pAb, LAPTM4B-N₁₋₉₉-pAb polyclonal antibodiesand LAPTM4B-N₁₋₉₉-mAb monoclonal antibody can be used to analyze LAPTM4Bprotein expression, intracellular localization, separation andpurification, and protein-protein interaction. They can also be used todetect the antibody and antigen of LAPTM4B in blood (FIG. 8). Moreover,LAPTM4B-EC2₂₃₂₋₂₄₁-pAb can inhibit cancer cell proliferation (FIG. 12),Tyr₂₈₅ phosphorylation of LAPTM4B protein (FIG. 15-B), and thephosphorylation and activation of signal molecules FAK (FIG. 16-A) andMAPK (FIG. 16-B). Therefore, all the monoclonal and polyclonalantibodies for SEQ ID No: 4 LAPTM4B-35 protein epitope are encompassedin this invention.

SEQ ID No: 8 is the promoter sequence of LAPTM4B gene. To study theregulation of LAPTM4B gene expression, the LAPTM4B gene promoter and theupstream sequence SEQ ID No: 8 are cloned. There are no typical CCAAT(TTGCGCAAT), TATA cassettes in LAPTM4B gene promoter region. But variousbinding sites of transcription factors exist in the upstream region ofLAPTM4B promoter, such as CREBP1/c-Jun, CEBP, PAX2/5/8, GATA, STAT,c-Ets-1, E2F, LYF-1, and c/v-Myb (FIG. 17A). These transcription factorsmay specifically regulate LAPTM4B expression in cells of varioustissues. The abnormal expression and activation of these transcriptionfactors in cancer cells possibly lead to an unbalanced expression ofLAPTM4B proteins. Moreover, there are two highly homologous repeatingsequences in the upstream domain of LAPTM4B promoter. It is worthwhileto study whether they are responsible to the regulation of LAPTM4Bexpression. A series of vectors consisting upstream region sequences ofLAPTM4B promoter with different lengths—promoter-5′ UTR-35 bp encodingregion—luciferase reporting gene is constructed, and these vectors areused to transfect into BEL7402 cells and HLE cells from humanhepatocellular carcinoma HCC. As shown in FIG. 17, the cells transfectedwith various vectors all show luciferase activity with variousintensities, indicating the transcription activities in these segments.The smallest fragment is a DNA segment (pGL-PF4) at about 38 bp in theupstream region of the transcription starting site. It possesses a basicpromoting activity and functions as LAPTM4 core promoter. The activityof pGL-PF4 transfectant in BEL7402 is 20% of the reference promoterSV40, while the activity is low in HLE, only 6% of SV40 activity, about⅓ of that in BEL7402. These data partially reflect the natural activityof LAPTM4B promoter in these two cell lines. It is consistent with theNorthern blot results, where mRNA expression is high in BEL-7402 cellline and low in HLE cell line. Additionally, pGL3-PF4 transfectantreveals dramatically different activities in these two cells. Itsactivity in BEL-7402 cells is 7 times higher than that in HLE cells.Apparently, different regulative mechanisms of LATPM4B genetranscription exist in BEL7402 and HLE cell lines.

In embodiments of this invention, the genome DNA is genotyped in orderto determine the relationship between different LATPM4B genotypes andsusceptibility of hepatocellular carcinoma. LATPM4B has two alleles,LATPM4B*1 and LATPM4B*2, i.e., SEQ ID No: 6, is derived by PCR cloning.As shown in FIG. 9, the difference between alleles *1 and *2 is the 19bp sequence in the first exon 5′ UTR. Allele *1 has only one suchsequence (nt 124˜142dup, while *2 contains two such sequences in atightly tandem arrangement (124-142dup, taking G at the transcriptionstarting site TSS as +1 numbering standard). The insertion of the 19-bpsequence would eliminate the stop codon in 5′UTR in the corresponding *1allele by a triplet shift. As a result, the open reading frame may beextended upstream by 53 amino acids at N terminus of the protein. Theencoded protein by SEQ ID No: 6 then should contain 370 amino acidresidues (SEQ ID No: 7). LATPM4B genotypes detected in human populationare *1/*1, *1/*2 and *2/*2, respectively (FIG. 10). Studies show thatthe risk of developing hepatocellular carcinoma for individuals withLATPM4B genotype *2/*2 is 2.89 times higher than individuals withnon-*2/*2 type (Table 2). However, LATPM4B genotype in patients withesophagus carcinoma shows no difference from the normal population(Table 3). This indicates that LATPM4B *2/*2 genotype correlatesespecially to susceptibility of hepatocellular carcinoma. As a result,LATPM4B allele LATPM4B*2 provided by this invention can be used as atarget to screen and diagnose people susceptible to hepatocellularcarcinoma or having a high risk to develop hepatocellular carcinoma.Particularly, using LATPM4B *2/*2 genotype as a target to screen highlysusceptible or high risk people can be more accurate. *1/*1, *1/*2 and*2/*2 of LATPM4B genotypes, LATPM4B*2 encoded proteins or theirantibodies, and/or LATPM4B extender and scavenger from human genome canall be used to screen people who are susceptible to hepatocellularcarcinoma or having a high risk to develop hepatocellular carcinoma.

The expression vectors containing sequences described in SEQ ID No: 1,2, 3, 6, 8, the transfection cell lines containing SEQ ID No: 1, 2, 3,6, 8 sequences, and the primers amplifying SEQ ID No: 1, 2, 3, 6, 8 areall encompassed by this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-A is the Northern Blot analysis profiles indicating thetranscriptive expression of the gene of this invention in normal humanliver, normal fetus liver and hepatocellular carcinoma tissues.

FIG. 1-B is the Northern Blot analysis spectrum indicating thetranscriptive expression of the gene of this invention in the humanhepatocellular carcinoma cell lines.

FIG. 1-C is a diagram showing the relationship of the expression levelof the gene of this invention in human hepatocellular carcinoma tissuesand the cancer differentiation status.

FIG. 2-A is a diagram of hybridization in situ of hepatocellularcarcinoma tissue. The LATPM4B mRNA in hepatocellular carcinoma noduleshows a strong positive staining.

FIG. 2-B is an immunohistochemical diagram, where LATPM4B protein inhepatocellular carcinoma nodule shows a strong positive staining.

FIG. 2-C is an immunocytochemical diagram, where LATPM4B protein isshown to exist in the transfected cells.

FIG. 3 presents a Western Blot analysis diagram, where the expressionspectra of LATPM4B-35 and LATPM4B-24 proteins encoded by the gene ofthis invention are shown in the tissues of normal liver (NL),hepatocellular carcinoma (HCC), and paired non-cancerous liver (PNL).

FIG. 4 shows a growth curve of the accelerated proliferation ofcDNA-transfected cells of this invention.

FIG. 5 shows a column diagram, where the DNA synthesis of LAPTM4BcDNA-transfected cells of this invention is increased.

FIG. 6 is a pie diagram showing an increase of cell numbers in S phasein cDNA-transfected cells of this invention (Flow cytometry analysis).

FIG. 7 shows the oncogenic effect of cDNA-transfected cells of thisinvention on mouse (SEQ ID No. 19).

FIG. 8 is a histogram showing the level of the antigen of this inventionin the serum of patients with hepatocellular carcinoma.

FIGS. 9 A and B shows schematic diagrams of the LAPTM4B promoter and itsfirst exon and the partial sequence (SEQ ID No. 19 and SEQ ID No. 20) ofLAPTM4B alleles of this invention.

FIG. 10 shows the genotypes distribution of LAPTM4B alleles of thisinvention in human population.

FIG. 11 is an immunohistochemical diagram of various cancer tissuesderived from epithelium.

FIG. 12 is a column diagram showing the inhibitory effect ofLAPTM4B-EC2-pAb antibody on proliferation of hepatocellular carcinomacells.

FIGS. 13-A, 13-B, 13-C, 13-D, and 13-E are the Western Blot analysisdiagrams showing respectively that the expressions of cyclin D1, cyclinE, c-Myc, c-Fos, and c-Jun of cDNA-transfected cells of this inventionare up-regulated.

FIGS. 14-A, 14-B, and 14-C are the analytical diagrams of the co-immunoprecipitation, revealing respectively the interactions of the geneproduct (LAPTM4B protein) of this invention with α 6 β 1 integrin andwith the epidermal growth factor receptor (EGFR).

FIGS. 15-A, 15-B, and 15-C are the analytical diagrams of theimmunoprecipitation, showing the Tyr phosphorylation of LAPTM4B proteinand the inhibitory effect of LAPTM4B-EC2-pAb on Tyr phosphorylation.

FIGS. 16-A and 16-B are the analytical diagrams of the co-immunoprecipitation showing respectively that LAPTM4B is involved in FAK-MAPKsignal transduction pathway.

FIG. 17 is a plot showing the transcriptive activity of variousfragments of LAPTM4B promoter of this invention (SEQ ID No. 8).

DETAILED DESCRIPTION OF THE INVENTION

Sources of Patients and Normal Control Group:

57 cases of hepatocellular carcinoma patients, 50 males and 7 females,ranged in age from 35-70. Their average age was 54±6.0. The tissuestested came from surgically excided specimens. The blood samples for thecontrol group were collected from 206 similarly aged people with nosymptoms and no cancer according to clinic tests and from 209 new bornbabies' umbilical veins.

109 esophagus cancer patients, 76 males and 33 females, ranged in agefrom 30-70. Their average age was 55±5.4. The test tissues came fromsurgically excided specimens. 116 people with no symptoms and no cancer,as determined by clinical examination, were selected as the controlgroup S. Their blood samples were taken for testing. All the sampleswere extracted to obtain genomic DNA.

Statistical Method

Chi-square (X²) measurement and single factor ANOVA variance were usedto treat and analyze the data.

Example 1 Northern Blot Analysis of LAPTM4B Expressions in Four Types ofLiver Tissues at Various Proliferation and Differentiation Status

Four types of liver tissues at various proliferation and differentiationstatus were chosen. They were from normal adult livers (NL, with verylittle proliferation and high differentiation), fetus livers (FL, atvigorous proliferation and low differentiation), hepatocellularcarcinoma (HCC, out of controlled proliferation and abnormaldifferentiation), and paired non-cancerous livers (PNL, generally is ofprecancerous stage in an active proliferation status). The Northern Blotanalysis was used to detect the transcription of gene in these tissues.RNA samples were extracted from 5 normal adult liver tissues freshlyobtained from surgical excision: 5 liver tissues from abortive fetus, 55HCC tissues, and 55 paired non-cancerous liver tissues. Afterelectrophoretic separation, they were transferred to a nylon film andhybridized by Dig labeled probe. The film was washed at 68° C. and thehybridization signals were developed according to the manual. Theresults are shown in FIG. 1. Band 1 was the sample from fetus livers.Band 2 was from the normal adult liver sample. Bands 3, 5, 7, and 9 werethe samples from HCC. Bands 4, 6, 8, 10 were from PNL tissues. Theresults show that the expression of LAPTM4B in various liver tissues hasthe following order: HCC tissue>PPNL tissue and fetus livertissue>normal adult liver tissue.

Example 2 Clonings of LAPTM4B Gene, Allele, and Promoter

2-1 LAPTM4B Gene Cloning

By using fluorescence differential display technique, an unknown genecDNA segment (LC27) was obtained from differential display spectrum infour types of human liver tissues in different proliferation anddifferentiation status, such as normal adult livers (NL), fetus livers(FL), cancerous livers (HCC), and paired non-cancerous liver (PNL). TheLC27 segment (426 bp) was elongated by splicing homogenous sequencesaccording to the EST to the 5′ direction, and followed by RACE (rapidamplification of cDNA ends) and the high temperature RT-PCR techniques.Two full-length cDNA sequences, i.e., SEQ ID No. 2 and 3, were produced,and then confirmed by sequencing and BLAST program analysis.

2-2. LAPTM4B Promoter Cloning

The sequence of upstream region of the first exon of LAPTM4B gene at 5′terminal was obtained by biological informatics, and primers F1 and R1were designed. Using human genomic DNA from HCC as the template, LAPTM4Bpromoter and the upstream sequence was obtained by PCR using PlatinumPfx DNA polymerase. After Xho I and hind III enzyme cutting, they wereinserted into pGL3-Basicvector to form pGL3-PF1, and its sequence wasdetermined (i.e., the test result see portion a of FIG. 16).

As shown in FIG. 17a , no typical CCAAT (TTGCGCAAT) and TATA boxes werefound in the LAPTM4B promoter sequence. In the upstream region ofLAPTM4B promoter, there are many binding sites for a variety oftranscription factors, such as CREBP1/c-Jun, CEBP, PAX2/5/8, GATA, STAT,c-Ets-1, E2F, LYF-1, and c/v-Myb. They may function on regulation ofLAPTM4B expression. In hepatocellular carcinoma, the abnormal expressionand activation of these transcription factors possibly lead to anunbalanced expression of LAPTM4B proteins. Moreover, the LAPTM4Bupstream region contains two highly homologous repeating sequences. Itis worthwhile to further study on whether they have any effect onLAPTM4B expression regulation.

2-3. Cloning and Sequence Analysis of LAPTM4B Alleles

2-3-1. DNA Separation

Genome DNA was extracted from blood lymphocytes or cancer tissue samplesfrom surgical excision of hepatocellular carcinoma and esophaguscarcinoma according to the standard phenol-chloroform method.

2-3-2. Cloning and Sequence Analysis of the Alleles

By using the same procedures for the promoter sequence cloning, twoprimers,

(SEQ ID No. 11 F1: 5′ GCGCTCGAGGCTCCAGGTGGAAGAGTGTGC 3′,inducing XhoI enzyme cutting site at 5′ terminal sequence as indicatedby underlining), and

(SEQ ID No. 16 R1: 5′ GCGAAGCTTGGACTTGGCCATGTGACCCG 3′,inducing XhoI enzyme cutting site at 5′ terminal sequence as indicatedby underlining), were designed and synthesized based on LAPTM4B genesequence SEQ ID No. 3. The promoter sequence and its anterior sequencein the first exon of LAPTM4B were then cloned from human genomic DNA byPCR. The pGL3-PF1 vectors constructed from various human genomic DNAwere sequenced to screen the LAPTM4B alleles. The original LAPTM4Bsequence was designated as LAPTM4B*1. The other one was designated asLAPTM4B*2, i.e., SEQ ID No. 6 in the sequence listings. FIG. 9(A) showsthe schematic diagrams of the LAPTM4B promoter and its first exon. Therectangle frame indicates the first exon, the black color arearepresents the encoding area, the white color is the non-coded area, andthe gray area shows a 19 bp DNA sequence. The horizontal linerepresenting promoter part and F1, F2, R1, and R2 are where the fourprimers are located. “A” in the start codon ATG is defined as +1 in thesequence. FIG. 9 (B) shows the partial sequences of the LAPTM4B allelesand their sequencing graphic spectra. The underlined part is a 19 bp DNAsequence.

The results reveal that LAPTM4B*1 contains one copied 19 bp DNA sequenceand LAPTM4B*2 has two copied 19 bp DNA sequences, which are linked inthe non-coded area (nt −33-−15) of the first exon of LAPTM4B*1.

The sequence analyses indicate that LAPTM4B*2 and LAPTM4B*1 possess thesame promoter. There is no difference in sequences between LAPTM4Balleles *1 and *2 promoters.

2-3-3. LAPTM4B Genotype Classification

E2 (5′ GCCGACTAGGGGACTGGCGGA 3′, SEQ ID No. 9) and R2 (5′CGAGAGCTCCGAGCTTCTGCC 3′, SEQ ID No. 10)primers were designed and synthesized. A partial sequence of the firstexon of LAPTM4B was amplified by PCR using templates of genomic DNA fromnormal people, hepatocellular carcinoma, and esophagus carcinomatissues. PCR conditions were as follows: 96° C. pre-denature for 5 min;94° C. for 30 s, 68° C. for 30 s, 72° C. for 1 min, 35 cycles; 72° C.for 5 min; then the PCR products were conducted to 2% Agarose gelelectrophoresis analysis.

FIG. 10 shows LAPTM4 gene *1/*1, *1/*2, and *2/*2 three types in humanpopulation.

Example 3 Construction of the Reporter Plasmids and Analysis of thePromoter Activity

A series of vectors, that contain the upstream sequences with variouslength of the LAPTM4B promoter, 5′UTR, the 35 bp encoding sequence inexon and the luciferase reporting gene, were constructed, i.e., theLAPTM4B gene promoter and the upstream sequence was cut by Xho I and IHind III enzyme and connected to pGL3-Basic vector to form pGL3-PF1, andidentified by sequencing. Then pGL3-PF1 was used as a template, primersF2, F3, and F4 vs. R1 were used to amplify by PCR, respectively, toconstruct vectors, pGL3-PF2, pGL3-PF3, and pGL3-PF4 which containpromoter segments with various lengths and luciferase gene. Theseconstructs were identified by sequencing.

The sequences of these primers are as follows:

F1: 5′GCGCTCGAGGCTCCAGGTGGAAGAGTGTGC 3 (nt −1341-−1321, SEQ ID No. 17)F2: 5′ GCGCTCGAGTAA AAACGCTGTGCCAGGCGT 3′ (nt −881-−861, SEQ ID No. 18)F3: 5′ CCGCTCGAGTACCGGAAGCACAGCGAGGAT 3′ (nt −558-−538, SEQ ID No. 13)F4: 5′ GCGCTCGAGAGTAGAAGGGAAGAAAATCGC 3′ (nt −38-−18, SEQ ID No. 14)R1: 5′ GCGAAGCTTGGACTTGGCCATGTGACCCG 3′ (nt 172-191, SEQ ID No. 15)

These vectors were used to transfect BEL-7402 cells and HLE cellsseparately and the promoter activities were measured. As shown in FIG.17b , the vector-transfected cells all have luciferase activities withdifferent intensities. pGL3-PF3 showed similar activity in both BEL-7402cells and HLE cells, which was about 27% of the SV40 promoter(pGL3-Promoter) activity. When comparing it with pGL3-PF4 activity,however, there was almost no difference in BEL-7402 cells. In HLE cells,pGL3-PF3 activity was 7 times higher than pGL3-PF4. As shown in FIG. 17a, pGL3-PF3 (−41˜−558) has many potential binding sites for transcriptionfactors. One or many of them, especially c-Ets-1, may play a regulatingrole in HLE cells and make the luciferase activities of pGL3-PF3 andpGL3-PF4 tranfectants remarkably different in HLE cells. The pGL3-PF3activity is higher than that of pGL3-PF1 and pGL3-PF2 in both BEL-7402and HLE cells, implying that some negatively regulatory factor exists.One or more of these negatively regulatory factors bind with thepromoter upstream target sequence (−558 upstream) to induce adownregulated LAPTM4B gene expression. This suppressive effect wasstronger in HLE cells than in 7402 cells. This means that HLE cells maycontain some factors that strongly suppress the expression of LAPTM4B.The Northern Blot analysis presented in FIG. 1-B also shows a lowexpression of LAPTM4B in HLE cells, supporting the above hypothesis. ThepGL3-PF2 vector contains two DNA repeating fragments (−41˜−328,−574˜−859), which is one more DNA fragment (−574˜−859) than pGL3-PF3.pGL3-PF3 exhibits higher activity than pGL3-PF2 in both cells. Thisresult indicates that the two repeating sequences negatively regulategene transcription. They have many potential binding sequences for thetranscription factors and provide two binding sites for each negativelyregulating factor. Since many transcription factors often form dimers,they have to bind with two target sequences to be able to function. Inthe case of pGL3-PF3, which can only provide one binding site, nofunction is shown. Since the pGL3-PF3 transfectant has a disinhibitoryeffect, its activity is higher than other vector transfectants.

Example 4 Western Blot Analysis of LAPTM4B Protein Expression

The tissue sample was placed on ice and cut into small pieces byscissors. 0.1 gram of wet tissue was selected and placed in a manuallyoperated homogenizer. 1 mL lysis buffer was added in each tube and themixture was thoroughly homogenized. The lysate was transferred to afresh tube and centrifuged at 4° C., 12 000 g for 10 min to remove thedebris. If cells are used, the cells in a culture dish were digestedwith 0.25% trypsin buffer, followed by two PBS rinses and centrifuged at500 g for 3 min. The cleared supernatant was collected, and the proteinsin the supernatant were separated by SDS-PAGE electrophoresis, and thentransferred to the NC membrane. The membrane was blocked at 4° C.overnight with 5% non-fat powdered milk in a TBS buffer containing 0.05%Tween 20. Then it was incubated with the rabbit polyclonal antibody,LAPTM4B-EC2 ₂₃₂₋₂₄₁-pAb (1:500 dilution) or mouse Anti-FLAG M2 monocloneantibody (Sigma, 1:750 dilution) at room temperature for 2 hours, andthen rinsed with TBS for three times. It was further incubated with aperoxidase-coupled second antibody (IgG), such as goat anti rabbit orgoat anti mouse (1:3000 dilution), for 2 hrs, followed by three rinseswith a TBS buffer (pH 8.0, containing 0.05% Tween 20). The last wash waswith a buffer containing no Tween 20. ECL (Santa Cruz) was used toexpose the positive bands (performed as manufacturer's instructions).When two antibodies were sequentially hybridized in one membrane, theECL exposed membrane was rinsed first with TBS followed by washing with30 mL TBS (2% SDS and 210 μL β-mercaptoethanol) for 30 min at roomtemperature. The 30 min TBS rinse removed the previous antibody and itssignal in the membrane, which then could be used for the secondhybridization. FIG. 3 shows that LAPTM4B-35 was over expressed in HCCtissues and HCC cell lines.

Example 5 Regulatory Effect of the Gene of this Invention on CellProliferation and the Malignant Phenotype of Cancer Cells asDemonstrated by a Full-Length cDNA Transfection

Using pGEMT-E2E7 plasmid as a template and the PCR method, a full lengthor partial cDNA, or the reading frame of LAPTM4B gene was amplified byPCR with primers A, or B and E, and the Pfx DNA polymerase. BamHI enzymecutting site (GGATCC) and ribosome binding site sequences (GCCACC) wereintroduced in primer A and B at 5′ terminal and EcoRI enzyme cuttingsite (GAATTC) was incorporated in the primer E. The amplified productsAE and BE were digested by restriction enzymes BamHI and EcoRI,purified, and ligated into pcDNA 3.0 vector. They were transformedconventionally to DH5 E. coli and the positive clone was selected, andthe constructed plasmid was sequenced for identifying. The constructedplasmids were named as pcDNA3/AE and -BE, respectively. pcDNA3/AEcontains a full-length ORF, while pcDNA3/BE contains the ORF startingfrom the second ATG to TAA. Compared with pcDNA3/AE, pcDNA3/BE-encodedprotein is missing 91 amino acids at the N terminal.

Mouse BHK, NIH3T3 cell lines and human hepatocellular carcinoma HLE cellline, in which the expression of LAPTM4B were all at very low level,were transfected by pcDNA3/AE or -BE, and clones that LAPTM4B expressionwere stable and high were selected. The total viable cell numbers weredetermined by the acidic phosphatase method and the cell growth curvewas plotted. The cell cycle was analyzed by the flow cytometry. Theexpression levels of cell cycle-regulating protein, including cyclin D1and Cyclin E, and proto-oncogene products, including c-Myc, c-Fos, andc-Jun (transcription factors for regulating cell proliferation) weremeasured by the Western Blot analysis. The results show that the cellproliferation was accelerated after being transfected by LAPTM4B-AEexpressive plasmid (FIG. 4, 5, 6). Expressions of cyclin D1, cyclin E,c-Myc, c-Fos, and c-Jun were also greatly increased (FIG. 13-A, B, C, D,E, respectively). But the dependence of growth on serum inLAPTM4B-35-overexpressed cells was greatly reduced (HLE-AE cellproliferation proceeded normally in 1% FCS, but HLE and HLE-MOCK cellsstop proliferation at the same condition). In the meantime, theanchorage-dependent cell growth of HLE-AE cells was clearly weakened.Large colonies of HLE-AE cells were formed in the soft agar, whichindicates that this gene participated in the regulation of cellproliferation and its over expression (activation) was related to thedysregulation of cell proliferation. Furthermore, the migratingcapability of HLE-AE cells was also enhanced (The HLE-AE cells thatmigrated through the membrane pores were increased from 1216±403.8 forthe control to 4082.5±748.8). Its capability to invade Matrigel was alsogreatly increased (from 25±12.73 cells for the control to 1325±424.26cells). The results show that LAPTM4B over expression promotes thedevelopment of cell malignant phenotype. On the contrary, BHK-BE,NIH3T3-BE, and HLE-BE cells transfected by LAPTM4B-BE expressive plasmidcould not form clones. They were all dead within three weeks. Theseresults demonstrate that LAPTM4B-24 plays antagonistic roles toLAPTM4B-35.

Example 6 Tumorigenic Effect of LAPTM4B cDNA-Transfected Cells on Mouse

Six-week old male mice were randomly selected and divided into threegroups: In the first control group, the mice were injected withphysiological saline. In the second control group, the mice wereinoculated with the pcDNA3 MOCK (no-load plasmid) transfected cells by.In the test group, all the mice were inoculated with pcDNA3/AE (aplasmid containing full-length cDNA) transfected NIH3T3 cells. Eachmouse was subcutaneously inoculated with 2×10⁶ cells. There were four tosix mice in each group. The mice were sacrificed after 21 daysinoculation and dissected. As shown in FIG. 7, two mice (half ofinoculated mice) in the test group developed a clearly moderatemalignant fibrosarcoma (A, B); the other two mice were identified aslymphatic tissue at the inoculated sites (C, D). In contrast, twelvemice in the two control groups showed no sign of tumor formation afterbeing inoculated for 86 days.

The results in Examples 4, 5, and 6 indicate that LAPTM4B may be a novelproto-oncogene.

Example 7 Primary Analysis of LAPTM4B Antigen in the Serum of Patientswith Hepatocellular Carcinoma by the ELISA Method

96 wells culture plates were coated with sera in various dilutions fromHCC patients and normal people by known agreement at 4° C. overnight.Each well was washed with 0.5% Tween-20 washing solution, and then 2%BSA was added for blocking at room temperature for 1 hour. ThenLAPTM4B-EC2-pAb antibody in various dilutions was added and incubatedfor 2 hours at room temperature. The goat anti-rabbit antibody labeledby horseradish peroxidase (1:1000 times dilution) was added after PBSwashing. After standing at room temperature for 2 hours and one PBSwashing, 1 g/mL o-phenyldiamine was added for 10-15 minutes to developcolor and H₂SO₄ was used to stop the reaction. The microtiter for enzymeanalysis was used to measure OD. at 490 nm and the antigen level wasestimated. The results are shown in FIG. 8. Clearly, the sera ofpatients with hepatocellular carcinoma contained higher level of LAPTM4Bantigen than that from normal people, indicating that LAPTM4B has apotential to become a new marker for hepatocellular carcinoma diagnosis.

Example 8 Functional Determination of LAPTM4B Protein in SignalTransduction by Co-Immunoprecipitation and Antibody Inhibition Analysis

The cell lysate was prepared according to the method in Example 4. Thefirst antibody was added to the supernatant. After 1 hour's shaking at4° C., 50 μL protein G-Agarose suspension was added and the mixture wasshaken at 4° C. for at least three hours or overnight. The immunocomplexprecipitate was collected after centrifuging at 12000 g for 20 seconds.The complex was re-suspended by adding 1 mL washing buffer I and shakenat 4° C. for 20 min. The mixture was centrifuged at 12000 g for 20seconds and the supernatant was removed carefully. This step wasrepeated once. Then the complex was re-suspended by adding washingbuffer II, shaken at 4° C. fro 20 min., and centrifuged at 12000 g for20 seconds. The supernatant was removed carefully. The last two stepswere repeated once. The complex was re-suspended by adding washingbuffer III, shaken at 4° C. fro 20 min, and followed by 12 000 gcentrifugation for 20 seconds. The supernatant was removed completely.50 μL 1×SDS loading buffer was added in the precipitate and the mixturewas boiling in 100° C. water bath for 5 min to denature and dissociatethe immunocomplex in the sample. After 12000 g centrifugation for 20second, the supernatant was removed and analyzed in SDS-PAGE apparatus.

BEL-7402 cell was preincubated for 0, 10, 20, and 40 min, respectively,on LN-1 substance in serum free medium. Co-immunoprecipitation wasperformed with LAPTM4B-EC2-pAb from the cell lysate. Theco-immunoprecipitates were respectively adsorbed by Protein G-Sephorose,centrifuged, and analyzed by 10% non-reductive SDS-PAGE. Then thephosphorylations of LAPTM4B, FAK and MAPK were analyzed separately bythe Western Blot with p-Tyr mAb.

BEL-7402 cells were preinoculated separately with LAPTM4B-EC2-pAb (15μg/mL) and anti-Glut2 (15 μL/mL) antibodies at 37° C. under 5% CO₂ for 2hrs, and then seeded on LN-1 substance and incubated for indicatingtime. Under the same conditions, the anti-Glut2 antibody treated cellsand no antibody treated cells were used as control. The cell lysate ineach group was analyzed by the Western Blot analysis with p-Tyr mAb. Theinhibitory effects of various antibodies on phosphorylation of LAPTM4Bwere analyzed. The results show that LAPTM4B-35 was phosphorylatedpeakly when human hepatocellular carcinoma BEL-7402 cells were attachedon laminin substrate. The phosphorylation of LAPTM4B-35 reached thehighest level in 10 min after cell attachment (FIG. 15-A). MeanwhileLAPTM4B-EC2-pAb could inhibit almost completely its phosphorylation(FIG. 15-B), while the anti-Glut2 (an antibody against a non-relatedplasma membrane protein Glut2) showed no such inhibitory effect (FIG.15-C). On contrary, LAPTM4B-24 cannot be phosphorylated. Thephosphorylation of LAPTM4B-35 Tyr₂₈₅ would form a binding site forsignal molecules that contain SH2 domain. In the meantime, LAPTM4B-35itself presents typical binding sites for signal molecules that containSH3 domain. Therefore, LAPTM4B-35 functions most likely as a veryimportant docking protein of molecules for signal transduction or aspecial organizer of membrane microdomain. It could recruit signalmolecules related inside or outside cells, so that to play pivotal rolesin signal transduction associated with cell proliferation,differentiation and apoptosis. Moreover, the attachment of humanhepatocellular carcinoma cells on laminin substrate can also cause Tyrphosphorylation of the cytoplasmic signal molecule FAK (FIG. 16-A), andthe LAPTM4B-EC2-pAb and anti-integron α 6 mAb against the epitope of theextracellular region of α6 both can prevent FAK phosphorylation withoutaffecting the expression level of FAK protein by preincubating with BEL7402 cells. Similarly, the attachment of BEL 7402 cells on lamininsubstrate can also induce Tyr phosphorylation of the signal moleculeMAPK (FIG. 16-B), and its phosphorylation can be inhibited bypreincubating cells with LAPTM4B-EC2-pAb without changing the expressionlevel of MAPK protein. These results indicate that the interactionbetween LAPTM4B-EC2 domain (the second extracellular region) andintegrin α6 subunit plays an important role in triggering FAK-MAPKsignaling pathway.

The results from Examples 4-8 suggest that LAPTM4B-35 can be potentialtargets of drugs for regulating cell proliferation, differentiation, andapoptosis.

Example 9 LAPTM4B Genotype Classification

LAPTM4B genotypes in genomic DNA from blood of normal individuals andpatients with hepatocellular carcinoma were detected by PCR. Two primerswere designed and synthesized according to the flanking sequence of 19bp DNA sequence in LAPTM4B gene sequence 3:

(SEQ ID No. 9) E2: 5′ GCCGACTAGGGGACTGGCGGA 3′ (SEQ ID No. 10) R2: 5′CGAGAGCTCCGAGCTTCTGCC 3′

The partial sequence of the first exon was amplified using genomic DNAas a template. PCR conditions were as follows: 96° C. pre-denature for 5min, 94° C. for 30 sec, 68° C. for 30 sec, 72° C. for 1 min, 35 cycles,72° C. extension for 5 min. PCR products were analyzed by 2% agarose gelelectrophoresis and the results are shown in FIG. 10. The lanes 1, 6,12, and 13 represent a 204 bp nucleotide segment in LAPTM4B*1/*1. Thelanes 5, 8, 9, 14, and 15 represent a 223 bp nucleotide segment inLAPTM4B*2/*2. The lanes 2, 3, 4, 7, 10, and 11 represent 204 bp and 223bp nucleotide segments in LAPTM4B*1/*2. Line M is the marker. Theresults reveals that in the homozygous gene pair of *1/*1 or *2/*2either the 204 bp or 223 bp DNA segment was amplified, while in *1/*2hybrid gene pair 204 bp and 223 bp DNA segments were both amplifiedsimultaneously. Therefore, the genotype of LAPTM4B in Chinese populationcan be classified as LAPTM4B*1/*1, *1/*2, and *2/*2 (FIG. 10).

Example 10 Frequency Distribution of LAPTM4B Genotypes and Alleles inNormal People and Patients with Hepatocellular Carcinoma

In one of the embodiments of the present invention, the occurrencefrequency of LAPTM4B genotypes in 209 normal Chinese and 57 patientswith hepatocellular carcinoma was analyzed and compared in Table 2. TheHardy-Weinberg equation was used to get the expectancy analysis. Thefrequency of LAPTM4B allele *1 and *2 from patients with hepatocellularcarcinoma differs significantly from that of normal people. Their ratiosare 0.5175: 0.6746 and 0.4825: 0.3254, respectively. The occurrencefrequencies of LAPTM4B allele *1 and *2 in a normal population are0.6746 and 0.3253, while the occurrence frequency of LAPTM4B allele *1and *2 in patients with hepatocellular carcinoma are 0.5175 and 0.4825.The occurrence frequency of genotype *1/*1 (p=0.029) and *2/*2 (p=0.003)in the group of hepatocellular carcinoma patient shows a significantstatistical difference from its control group. In the hepatocellularcarcinoma patient group, only 29.8% is of *1/*1, while in the normalcontrol group, 45.93% is of *1/*1. The occurrence frequency of *2/*2genotype in the hepatocellular carcinoma patient group is 26.32% ascompared to 11.01% in the control group, therefore its occurrencefrequency is increased significantly (p<0.01). The analysis shows thatthe risk suffering from HCC of individuals in *2/*2 genotype of is 2.89times greater than that in other genotype in developing hepatocellularcarcinoma. Thus, the LAPTM4B*2/*2 genotype is correlated with thesusceptibility of developing hepatocellular carcinoma.

As shown in Table 3, patients with different LAPTM4B genotypes did notshow any differences in hepatocellular carcinoma Grade, stage, or HBVinfection. 83.3% of the HCC patients have a positive HBV.

TABLE 2 Distribution of LAPTM4B genotype in hepatocellular carcinomapatients and normal population N (%) Hepatocellular Control B carcinomagroup (n = 209) (n = 57) P Value LAPTM4B genotype *1/*1 96 (45.93) 17(29.82) 0.029^(a) *1/*2 90 (43.06) 25 (43.86) 0.914 *2/*2 23 (11.01) 15(26.32) 0.003^(b) Frequency of alleles *1 0.6746 0.5175 *2 0.3254 0.4825^(a)OR: 0.500, 95% CI: 0.267-0.939; ^(b)OR: 2.888, 95% CI: 1.390-6.003(OR risk suffering HCC, and 95% CI is confidence interval)

TABLE 3 Clinical data of the hepatocellular carcinoma patients used inLAPTM4B genotype classification LAPTM4B Genotype *1/*1 *1/*2 *2/*2 PValue Total number 17 25 15 NS Males 14 24 12 Females 3 1 3 Cancer GradeG1 0 2 0 NS G2 1 4 8 G3 7 7 4 G4 9 12 3 Cancer stage I 0 0 0 NS II 5 8 5III 4 7 3 IV 8 10 7 HBV Infection Negative 1 4 4 NS Positive 13 16 10 Nodiagnosis 3 5 1 NS: No significant difference

Example 11 Frequencies of Genotype and Allele in Patients with EsophagusCarcinoma

To study if the LAPTM4B genotype is related to the susceptibility ofdeveloping other cancers, the genomic DNA from blood of 116 normalpeople and 109 patients with esophagus carcinoma from the same locationwere analyzed. As shown in Table 4, LAPTM4B genotype of patients withesophagus carcinoma is no significant different from control group ofthe normal population. LAPTM4B alleles are not related with thesusceptibility of developing esophagus cancer.

TABLE 4 Distributions of LAPTM4B genotypes of patients with esophaguscarcinoma and normal population N (%) Control Control Esophagus group Bgroup S carcinoma (n = 209) (n = 116) (n = 109) P Value LAPTM4B genotype*1/*1 96 (45.93) 52 (44.83) 49 (44.95) >0.05 *1/*2 90 (43.06) 49 (42.24)48 (44.04) >0.05 *2/*2 23 (11.01) 15 (12.93) 12 (11.01) >0.05 Allelefrequency *1 0.6746 0.6595 0.6697 *2 0.3254 0.3405 0.3303

Example 12 LAPTM4B-35 Expression in Some Epithelium Sourced Cancers

The relationship between the LAPTM4B-35 protein expression and othercancers was studied by an immunohistochemical method. The fixedspecimens from esophagus cancer, breast cancer, lung cancer, stomachcancer, colon cancer, and rectal cancer positive tissues and thenegative control noncancerous tissues were obtained from surgicalexcision and treated according to the following steps:

1. Specimen dewaxing by xylene

2. Katocromy with different concentrations of ethanol,100%-95%-90%-80%-70%. H₂O₂ was used to remove endogenous peroxidase

3. Antigen repairing by sodium citrate

4. PBS rinse twice

5. Normal goat serum blocking

6. Keep LAPTM4B-N₁₋₉₉pAb at 37° C. for 1 hour

7. PBS rinse three times

8. Keep HRP labeled goat anti-rabbit antibody at 37° C. for 1 hour

9. PBS rinse three times

10. Develop color by DAB

11. Nuclear retaining with hematoxylin

12. Ascending dehydration by ethanol at different concentrations(70%-80%-90%-95%-100%

13. Mounting

As shown in FIG. 11, the 11-A indicates a normal esophagus tissue(Negative), B is an esophagus cancer tissue (Negative), C is a normalbreast tissue (Negative), D is the breast cancer tissue (Positive), E isa normal lung tissue (Negative), F is a lung cancer tissue (Positive), Gis a normal stomach tissue (Negative), and H is a stomach cancer tissue(Positive). As can be seen from the figures, LAPTM4B was clearlyexpressed in lung cancer, stomach cancer, and breast cancer tissues,while it was not expressed clearly in esophagus cancer and largeintestine cancer.

INDUSTRIAL APPLICATIONS

The proteins encoded by LAPTM4B gene in this invention could be possiblyused as new markers in early diagnosis of some cancers. By using thewidely applied ELISA method in clinical tests, and the prepared relatedtesting reagent kits, the efficiency and the accuracy of the earlydiagnosis of cancers, especially the primary hepatocellular carcinoma,can be improved.

LAPTM4B gene can be used as target gene in the cancer treatment.Suppressing LAPTM4B-35 expression and promoting LAPTM4B-24 expressioncould inhibit the growth of hepatocellular carcinoma cells, reversemalignancy phenotype or delay its development. For example, theexpression products of LAPTM4B gene, LAPTM4B-35 could be inhibited bythe newly developed siRNA interference technology. Furthermore,LAPTM4B-BE-cDNA could be recombinated in the engineered virus expressionvector and be used in antitumor gene therapy through an up-regulation ofLAPTM4B-24 expression. LAPTM4B-35 protein could also be used as a newtarget for pharmaceutical treatment. Since LAPTM4B-35 protein canfunction as an assembling platform for complex of cell signaltransduction molecules, and it contains a number of binding sites forsignal molecules, there is a great potential to develop various newmedicines with LAPTM4B protein as targets. Moreover, this invention hasinitially demonstrated that LAPTM4B-EC2-pAb antibody can inhibit tumorcell proliferation and block its signal transduction. Based on thediscovery in this invention, further studies can be pursued on thepossibility of using antibody to inhibit hepatocellular carcinoma andsome other cancer development. After a better understanding on theeffect, a humanized soluble single chain antibody could be developed forclinical treatment on HCC patients. Peptide vaccines could also bedeveloped. If the vaccines can be successfully made, it will not onlyhelp cure hepatocellular carcinoma and some other cancer, but alsoprevent cancerogenesis in the high risk population. In summary, many newanticancer approaches can be developed based on the embodiments of thisinvention. As important supplements for treatments of hepatocellularcarcinoma and other cancers, this invention will help increase the curerates of hepatocellular carcinoma and other cancers. This project wouldgenerate a significantly great impact on human society.

In specific embodiments, LAPTM4B genotype of genomice DNA is genotyped.The relationship of various genotypes with the susceptibility tohepatocellular carcinoma as well as with other cancers s isinvestigated. It is discovered that one of the genotypes, LAPTM4B*2/*2,is correlated closely to hepatocellular carcinoma susceptibility. As aresult, it provides a new and accurate criterion for screening peoplewho are susceptible to primary hepatocellular carcinoma in the high riskpopulation. It is of important significance to the assessment andprevention of high risk population from developing hepatocellularcarcinoma.

The invention claimed is:
 1. An in vitro method of detecting LAPTM4B-35protein in a human tissue, blood or serum sample, comprising (a)obtaining a human tissue, blood, or serum sample; (b) detecting thelevel of LAPTM4B-35 protein in said sample, wherein the amino acidsequence of LAPTM4B-35 protein comprises the amino acid sequence of SEQID NO:
 4. 2. The method according to claim 1, further comprisingdetecting the level of LAPTM4B-24 protein in said sample, wherein theamino acid sequence of LAPTM4B-24 protein consists of the amino acidsequence of SEQ ID NO:
 5. 3. The method according to claim 2, furthercomprising the step of determining the ratio of the level of LAPTM4B-35protein to the level of LAPTM4B-24.
 4. The method according to claim 1,wherein the sample is a tissue selected from the group consisting ofliver, stomach, breast, lung and prostate.
 5. The method according toclaim 1, wherein the sample is from a patient at risk of cancer.